Immunofluorescence analysis of Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 555 (Product # A31570) was performed using HeLa cells stained with alpha Tubulin (236-10501) Mouse Monoclonal Antibody (Product # A11126). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with mouse primary antibody (1:250 dilution) for 3 hours at room temperature. Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 555 was used at concentration of 4µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Alexa Fluor® 488 Phalloidin (A12379, 1:300) (Panel c: red). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Donkey / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 555|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||4 µg/ml|
|Immunofluorescence (IF)||1-10 µg/mL|
|Immunohistochemistry (IHC)||1-10 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
To minimize cross-reactivity, these donkey anti-mouse IgG whole antibodies have been affinity-purified and show minimum cross-reactivity to bovine, chicken, goat, guinea pig, hamster, horse, human, rabbit, rat, and sheep serum proteins. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there may be the presence of endogenous immunoglobulins.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 555 dye is a bright, orange-fluorescent dye with excitation ideally suited to the 555 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 555 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 555 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.
Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.
We offer an extensive line of Invitrogen™ secondary antibody conjugates with well-characterized specificity and labeled with a wide selection of premium fluorescent dyes, including Invitrogen™ Alexa Fluor™ fluorescent dyes. Fluorescent secondary antibody conjugates are useful in the detection, sorting, or purification of its specified target and ideal for fluorescence microscopy and confocal laser scanning microscopy, flow cytometry, and fluorescent western detection. The breadth of fluorescent markers we offer allows our reagents to be tailored to almost any fluorescent detection system.
Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments, and monovalent Fab fragments. Because of the high degree of conservation in the structure of many immunoglobulin domains, most class-specific secondary antibodies must be affinity-purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins.
Our secondary antibody conjugates are most commonly prepared by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (e.g., immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents. In the first round of purification, whole immunoglobulins binding to the immunizing antibody are recovered and mainly consist of the ~150-kDa IgG class. Further purification, for example, with Protein A or G, removes all unwanted immunoglobulin classes except the affinity-purified antibodies that react with the target-specific immunoglobulin heavy and/or light chains.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex.
A-31570 was used in immunohistochemistry - frozen section to investigate the role of MBD3/NuRD in neurogenesis.
|Knock E,Pereira J,Lombard PD,Dimond A,Leaford D,Livesey FJ,Hendrich B||Neural development (10:null)||2015|
Foxg1 has an essential role in postnatal development of the dentate gyrus.
A-31570 was used in immunohistochemistry - frozen section to determine the function of Foxg1 in postnatal dentate gyrus neurogenesis.
|Tian C,Gong Y,Yang Y,Shen W,Wang K,Liu J,Xu B,Zhao J,Zhao C||The Journal of neuroscience : the official journal of the Society for Neuroscience (32:2931)||2012|
|Not Applicable||Not Cited||Generation of induced pluripotent stem cells using recombinant proteins.||Zhou H,Wu S,Joo JY,Zhu S,Han DW,Lin T,Trauger S,Bien G,Yao S,Zhu Y,Siuzdak G,Schöler HR,Duan L,Ding S||Cell stem cell (4:381)||2009|
|Not Applicable||Not Cited||Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques.||Estes JD,Gordon SN,Zeng M,Chahroudi AM,Dunham RM,Staprans SI,Reilly CS,Silvestri G,Haase AT||Journal of immunology (Baltimore, Md. : 1950) (180:6798)||2008|
|Not Applicable||Not Cited||G-CSF rescues the memory impairment of animal models of Alzheimer's disease.||Tsai KJ,Tsai YC,Shen CK||The Journal of experimental medicine (204:1273)||2007|
|Not Applicable||Not Cited||TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain.||Urra S,Escudero CA,Ramos P,Lisbona F,Allende E,Covarrubias P,Parraguez JI,Zampieri N,Chao MV,Annaert W,Bronfman FC||The Journal of biological chemistry (282:7606)||2007|
|Not Applicable||Not Cited||Activation of NF-kappa B by the human T cell leukemia virus type I Tax oncoprotein is associated with ubiquitin-dependent relocalization of I kappa B kinase.||Harhaj NS,Sun SC,Harhaj EW||The Journal of biological chemistry (282:4185)||2007|
|Not Applicable||Not Cited||Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11.||Andersen OM,Schmidt V,Spoelgen R,Gliemann J,Behlke J,Galatis D,McKinstry WJ,Parker MW,Masters CL,Hyman BT,Cappai R,Willnow TE||Biochemistry (45:2618)||2006|
|Not Applicable||Not Cited||TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.||James D,Levine AJ,Besser D,Hemmati-Brivanlou A||Development (Cambridge, England) (132:1273)||2005|
|Not Applicable||Not Cited||Multiplex detection of RNA expression in Drosophila embryos.||Kosman D,Mizutani CM,Lemons D,Cox WG,McGinnis W,Bier E||Science (New York, N.Y.) (305:null)||2004|